Apoptosis is a strictly regulated process of programmed cell death in normal cells. Defects in the regulation can be related to specific disease conditions. Excessive apoptosis may be related to atrophy while insufficient apoptosis may lead to uncontrolled cell proliferation and cancer. Phospholipid phosphatidylserine (PS) is translocated from the cytoplasm to the plasma membrane upon apoptosis. Annexin V is known to bind PS and the degree of its binding to cells is therefore a good marker of apoptosis (Pigault, C., et al., J. Mol. Biol. 236, 199-208, 1994).
One common method for determination of apotosis is to incubate cells from a subject with labelled Annexin V for measuring of PS binding on the cell surface. Flow cytometry (i.e. FASC analysis) can be used for analysing cell surface proteins including cell binding proteins (Koopman et al., Blood 84, 1415-1420, 1994). Flow cytometry is a quantitative method in which the signals are related to individual cells. There are also ELISA assays with wells coated with anti Annexin V antibodies for measuring Annexin V levels in lysed cell samples. In this case, the comparative quantitation of signal between samples relies on equal cell number without normalization. Ensuring equal sample loading is difficult. Accuracy of results relies on total protein quantification and pipetting. Another possibility for measuring protein binding to cells is to grow cells in multiwell plates, incubate cells with labelled binder, wash to remove access binder and finally measure the signals from bound protein in each well. Equal number of cells in each well is difficult to control, since cell counting and pipetting is a manual procedure involves a risk of variation number of cells seeded in each well. Moreover, the cells may grow at different rate in each well until start of incubation of binder.